Mutant human insulin receptor DNA

ABSTRACT

A mutant human insulin receptor DNA, wherein the base sequence encoding Thr 831  in human insulin receptor DNA has been replaced by a base sequence encoding Ala and/or the base sequence encoding Tyr 1334  therein has been replaced by a base sequence encoding Cys; a fragment of the mutant human insulin receptor DNA containing the mutation part(s); a diagnostic probe for non-insulin-dependent diabetes mellitus comprising this fragment; and a diagnostic drug for non-insulin-dependent diabetes mellitus containing this fragment.

This application is a 371 of PCT/JP95/00906, filed May 12, 1995.

1. Technical Field

This invention relates to abnormalities in the insulin receptorstructure gene in non-insulin-dependent diabetes mellitus (hereinafterreferred to simply as NIDDM). More particularly, it relates to mutanthuman insulin receptor DNAs having mutations at specific sites andfragments thereof.

2. Background Art

NIDDM, which is a genetic disease with one of the highest incidence inmankind today, is induced by several gene abnormalities combined withenvironmental factors such as obesity, stress and aging. In Japan, thenumber of patients with NIDDM is estimated to be about 5,000,000. NIDDMhas been recently designated as one of the four most serious diseasesfollowing cancer, cerebral stroke and heart infarction. Thus there hasbeen an urgent need to establish an effective countermeasure for NIDDM.In general, the symptoms of NIDDM can be frequently ameliorated by diettherapy and kinesitherapy. Therefore, if possible, it is best todiagnose NIDDM at an early stage. Presently, in order to diagnose NIDDMat an early stage, troublesome examinations, such as OGTT, must beconducted. OGTT comprises orally administering 75 g of glucose to apatient in a fasting state, collecting the blood at intervals of 30minutes and measuring the blood sugar level at two hour intervals.Therefore, a more convenient and reliable diagnostic method is neededfor early detection and prevention of NIDDM.

Although no gene has been found to be responsible for the onset ofNIDDM, it is assumed that genes of insulin function mechanism-relatingfactors or genes of insulin secretion-relating factors may be candidategenes for NIDDM. It is known that the factors relating to insulinfunction involve insulin receptor, insulin receptor substrate (IRS-1),glucose transporter type 4, etc., while the factors relating to insulinsecretion involve glucose transporter type 2, glucokinase, chondriogene,etc. Although attempts were made to detect abnormalities in the lattertwo genes in association with NIDDM, the abnormality ratio was onlyaround 1% in each case (Interim Report in 1993, Onset Mechanism Group,Research and Study Project on Diabetes, Ministry of Health and Welfare).

Insulin acts upon the target cell by binding to the insulin receptorlocated on the cell membranae. Insulin resistance is often observed inthe early stages of NIDDM (Taylor, S. I. Diabetes 41:1473-1490, 1992).Based on these facts, it has been speculated that the insulin receptormight be the gene responsible for the onset of NIDDM. Abnormalities inthe insulin receptor would result in a high insulin resistance and thusinduce severe diabetes accompanied by hyperinsulinemia. However, such aphenomenon is scarcely observed in NIDDM cases. Thus, abnormalities inthe insulin receptor have not been related to NIDDM.

In recent years, a number of insulin receptor abnormalities have beendiscovered by others including the present inventors. It has been foundthat the examination data and symptoms of patients vary widely dependingon the mutation type (M. Taira et al., Science 245:63-66, 1989; F.Shimada et al., Lancet 335:1179-1181, 1990). Thus, it is quite possiblethat insulin receptor gene abnormalities may partially participate inthe onset of NIDDM. However, no attempt has been made so far tosystematically detect the insulin receptor gene abnormalities on a largescale in association with NIDDM. In addition, the particular locationsof the gene abnormalities are still unknown.

Under these circumstances, the present inventors have preparedchromosomal DNAs from the blood of typical NIDDM Japanese patients andstudied the base sequences of insulin receptor DNAs in order to revealthe relationship between human insulin receptor gene abnormalities andNIDDM. As a result, they have found out that a quantitative abnormalityis observed at a significant frequency in the patients with NIDDM, thuscompleting the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophorogram which shows part of the base sequence ofthe exon 13 of the insulin receptor β-subunit of five patients withNIDDM.

FIG. 2 is an electrophorogram which shows part of the base sequence ofthe exon 22 of the insulin receptor β-subunit of eight patients withNIDDM.

FIG. 3 shows insulin binding (A) and receptor tyrosine kinase activity(B,C) of wild type (IR^(WT)) and the mutant (IR^(A831)) receptors. (A)Confluent CHO cells expressing IR^(WT) (clone No. 12: ∘ - - - ∘, cloneNo. 21: ∘ - - - ∘) or IR^(A831) (clone No. 10: Δ - - - Δ, clone No. 17:Δ - - - Δ) were incubated with ¹²⁵ I-insulin of various concentrationsat 4° C. for 12 hours. The radioactivity of the bound ¹²⁵ I-insulin wasmeasured and plotted in accordance with the method of Scatchard. (B)Insulin-dependent receptor autophosphorylating activity was shown as theamount of ³² P incorporated into the insulin receptor β-subunit with theuse of the same cell lines as those employed in (A). (C) Anelectrophorogram of the immunoblotting of insulin-dependent tyrosinephosphorylations of IRS-1 and IR β-subunits measured with ananti-phospho-tyrosine antibody with the use of the same cell lines asthose employed in (A).

FIG. 4 shows insulin-induced complex formation of the normal insulinreceptor and two artificially mutated insulin receptors (IR^(A831) andIR^(C1334)) and the p85 subunit of PI 3-kinase. The p85 subunit of PI3-kinase was transiently co-expressed with the wild type IR (IR^(WT)) orthe mutant IRs (IR^(A831) and IR^(C1334)) in COS cells, respectively. Inthe presence of 10⁻⁷ M of insulin, the cell lysates wereimmunoprecipitated by an anti-β-subunit of IR (α-IRβ) or anti-p85 (αp85)antibodies. (A) and (B) are electrophorograms obtained by immunoblottingthe immunoprecipitates with (A) an anti-phosphotyrosine antibody (αPY)or (B) an anti-α-subunit of IR antibody (αIRα).

FIG. 5 shows analysis on the relationship between the mutation and theonset of NIDDM in a pedigree with the mutant IR^(C1334). (A) Familialanalysis of TAC (Tyr¹³³⁴)→TGC (Cys¹³³⁴) replacement. Squares and circlesstand respectively for male and female. Closed symbols stand forpatients with NIDDM, the arrow shows the proband and slashed symbolsstand for deceased. (B) Allele-specific hybridization. PCR productsoriginating from the insulin receptor exon 22 of the genomic DNAs ofthree patients and a normal subject were separated in an agarose gel andtransferred onto a nitrocellulose filter. After hybridizing with a ³²P-labeled oligonucleotide probe specific to the mutant allele, thefilter was autoradio-graphed. The IR^(C1334) mutation (a 346 bpfragment) was identified in one allele of the proband (Y. Y.) but not ina brother (T. S.) and a sister (S. K.).

FIG. 6 shows an analysis of the relationship between the mutation andthe onset of NIDDM in a pedigree with the mutant IR^(A831). (A) Familialanalysis of ACG (Thr⁸³¹)→GCG (Ala⁸³¹) replacement. The IR^(A831)mutation was identified in one allele of the proband (Yh. T.), twobrothers (I. T. and Yo. T.) and a sister (T. O.). The arrow shows theproband, squares and circles stand respectively for male and female.Closed symbols stand for patients with NIDDM, shaded symbols stand forIGT (impaired glucose tolerance) and slashed symbols stand for deceased.(B) An electrophorogram showing the detection of IR^(A831) by therestriction enzyme digestion. The IR^(A831) mutation in the PCR fragmentof the exon 13 cleaved a cleavage site specific for the restrictionenzyme CfoI. The CfoI-digestion product of the mutated PCR fragment (332bp) resulted in the appearance of two bands (102 bp and 220 bp), whilethis cleavage was prevented in the wild type.

FIG. 7 shows DNA (SEQ ID NO:1) (No. 1) encoding human insulin receptor.

FIG. 8 shows DNA (No. 2) encoding human insulin receptor.

FIG. 9 shows DNA (No. 3) encoding human insulin receptor.

FIG. 10 shows DNA (No. 4) encoding human insulin receptor, wherein themutation part (the amino acid at the 831-position) is boxed.

FIG. 11 shows DNA (No. 5) encoding human insulin receptor, wherein themutation part (the amino acid at the 1334-position) is boxed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a mutant human insulin receptor DNA,wherein the base sequence encoding Thr⁸³¹ in human insulin receptor DNAhas been replaced by a base sequence encoding Ala and/or the basesequence encoding Tyr¹³³⁴ therein has been replaced by a base sequenceencoding Cys, or a fragment of this mutant human insulin receptor DNAcontaining the mutation part(s).

The insulin receptor is a biomembrane receptor which specifically bindsto insulin and thus transmits the information to the inside of cells. Itconsists of two α chains (735 residues, molecular weight: 84, 214) andtwo β chains (620 residues, molecular weight: 69, 700). The insulinreceptor gene consists of 22 exons among which 11 exons encode the αsubunits while other 11 exons encode the β-subunits (S. Seino et al.,Proc. Natl. Acad. Sci. USA 86:114-118, 1989). The base sequence of humaninsulin receptor and the amino acid sequence corresponding thereto areshown in SEQ ID NO:1 in Sequence Listing. The mutant human insulinreceptor DNA of the present invention is one wherein the base sequence(ACG) encoding Thr⁸³¹ in the exon 13 of the β-subunit of human insulinreceptor DNA has been replaced by a base sequence (GCG) encoding Alaand/or the base sequence (TAC) encoding Tyr¹³³⁴ in the exon 22 of theβ-subunit has been replaced by a base sequence (TGC) encoding Cys, and afragment of said mutant human insulin receptor DNA containing themutation. The mutation parts of the mutant human insulin receptor of thepresent invention are boxed in the sequences shown in FIGS. 7 to 11identical with the sequence represented by SEQ ID NO:1 in SequenceListing.

The mutant human insulin receptor DNA of the present invention isobtained by amplifying all of the 22 exon genes of the insulin receptorof 51 typical Japanese patients with NIDDM by PCR (polymerase chainreaction) and inserting the PCR products thus amplified into the pUC19vector followed by DNA sequencing. As the result of the DNA sequencing,three patients had a heterozygous missense mutation Thr⁸³¹ →Ala⁸³¹ inthe exon 13 of the β-subunit and one patient had a heterozygous missensemutation Tyr¹³³⁴ →Cys¹³³⁴ in the exon 22 of the β-subunit.

The amino acid replacement observed in the former mutation (Thr⁸³¹→Ala⁸³¹) has never been reported hitherto. Although this mutation wasinduced on cDNA and forced to express in animal cells in order toanalyze the receptor function, no remarkable disorder in the receptorfunction was observed. Furthermore, this mutation was not observed in272 healthy volunteers. Thus statistical analyses indicate that thismutation relates to the onset of NIDDM. To further examine therelationship between this mutation (IR⁸³¹) and the onset of NIDDM, thedata on a family having the mutant IR^(A831) were analyzed. As a result,it is strongly suggested that the heterozygous mutation of IR^(A831)would cause the onset of NIDDM.

Similarly, no remarkable disorder in the receptor function is observedin association with the latter mutation (Tyr¹³³⁴ →Cys¹³³⁴). As theresult of analyses of a family, it was considered that this mutationdoes not relate to the onset of NIDDM. However, the insulin receptorhaving this mutation cannot bind to PI 3-kinase and the presentinventors have recently proven that PI 3-kinase participates in theinsulin signal transmission, particularly, translocation of glucosetransporter (F. Kanai et al., Biochem. Biophys. Res. Commun.195:762-768, 1993). Accordingly, it is suggested that this mutationmight relate to the onset of NIDDM.

The mutant human insulin receptor DNA of the present invention amountsto at least 6% of the quantitative mutations of the genes responsiblefor the onset of NIDDM, which is a level significant enough to ensurethe application thereof to gene diagnosis of NIDDM. There are a numberof genes participating in the onset of NIDDM and mutations in each ofthese gene have hot spots. It is assumed that several hot spots,including the mutation parts of the above-mention as well as the presentinvention, would be considered major ones. Supposing that othermutations participating in the onset of NIDDM could be clarified, thecombined use thereof with the mutant human insulin receptor DNA of thepresent invention or its fragment can provide a convenient and reliablemethod of gene diagnosis for NIDDM. It is expected, furthermore, thatthe early diagnosis of NIDDM with the combined use of this genediagnosis and the conventional diagnostic methods largely contributes tothe appropriate prevention and treatment of NIDDM.

The mutant human insulin receptor DNA of the present invention, itsfragment, DNAs complementary thereto and their fragments, are useful asa diagnostic probe to be used in the above-mentioned diagnosis. Thefollowing DNA fragments are usable as the diagnostic probe for NIDDMaccording to the present invention:

(a) a mutant human insulin receptor DNA fragment wherein the basesequence (ACG) encoding Thr⁸³¹ in the exon 13 of the β-subunit of humaninsulin receptor DNA has been replaced by a base sequence (GCG) encodingAla;

(b) a mutant human insulin receptor DNA fragment wherein the basesequence (TAC) encoding Tyr¹³³⁴ in the exon 22 of the β-subunit of humaninsulin receptor DNA has been replaced by a base sequence (TGC) encodingCys; and

(c) a DNA fragment which is complementary to the mutant human insulinreceptor DNA fragment of the above (a) or (b).

Such a DNA fragment for a diagnostic probe is generally composed of upto about 100 bases containing the above-mentioned mutation part,preferably from 10 to 50 bases containing the above-mentioned mutationpart and still preferably from 10 to 30 bases containing theabove-mentioned mutation part.

The DNA or DNA fragment of the present invention can be synthesized inaccordance with the base sequence of the present invention by using anautomatic DNA synthesizer with the use of, for example, the solid phasemethod performed on a support such as silica.

The above-mentioned gene diagnosis according to the present invention isnot particularly restricted in procedures, etc. and various methods maybe selected therefor over a wide range, so long as it aims at detectingthe above-mentioned specific mutation(s) characterized by the presentinvention. Since the gene mutations to be detected by the presentinvention have been clarified and specified, those skilled in the artcan easily select appropriate methods therefor in accordance with thedisclosure of the present invention.

For example, this method can be established by analyzing the basesequence of a specific site as defined above, which falls within thescope of the present invention as a matter of course. It therefor seemsreasonable to employ Southern hybridization or dot hybridization (eachdescribed in Southern, E. M., J. Mol. Biol., 98:503-517, 1975). It ispreferable to use a combination of PCR with a gene amplificationprocedure, since highly sensitive and accurate detection can beconveniently and easily performed with the use of a small amount of DNAspecimen. Examples of such a combination include PCR-RFLP (restrictionfragment length polymorphism) analysis, PCR-single strand polymorphismanalysis (Orita, M., Iwahata, H. Kanazawa, H., Hayashi, K. and Sekiya,T., Proc. Natl. Acad. Sci., USA, 86:2766-2770, 1989), PCR-SSO (specificsequence oligonucleotide) method, PCR-ASO (allele specific oligomer)nucleotide method with the use of dot hybridization (Saiki, R. K.,Bugawan, T. L., Horn, G. T., Mullis, K. B. and Erich, H. A, Nature,324:163-166, 1986), etc.

In the present invention, it is particularly preferable to use the RFLPanalysis and/or allele specific hybridization method from the viewpointof convenience. This detection method will be described in greaterdetail.

Various operations employed in the detection method of the presentinvention (for example, chemical synthesis of partial DNA, enzymatictreatments for the cleavage, deletion, addition or binding of DNA,isolation, purification, replication and selection of DNA, etc.) can beeach carried out in the conventional manner Bunshi Idengaku Jikken-ho(Experimental Methods for Molecular Genetics), Kyoritsu Shuppan, 1983;PCR Technology, Takara Shuzo, 1990!. For example, DNA can be isolatedand purified by agarose gel electrophoresis, etc. DNA can be sequencedby the dideoxy sequencing method (Sanger, F., Nicklen, S. and Coulson,A. R., Proc. Natl. Acad. Sci., USA, 74:5463-5467, 1977), Maxam-Gilbertmethod (Maxam, A. M. and Gilbert, W., Method in Enzymology, 65:499-560,1980), etc. Alternatively, DNA sequencing can be easily carried out byusing marketed sequence kits, etc. Also, PCR for amplifying a specificregion of DNA can be carried out in accordance with the conventionalmethod (see, for example, Saiki, R. K., Scharf, S., Faloona, F. A.,Mullis, K. B., Horn, G. T., Erlich, H. A. and Arnheim, N., Science,230:1350-1354, 1985). These fundamental operations are employed in, forexample, the references cited herein which are provided by way ofreference similar to the Examples presented hereinbelow.

The genomic DNA to be assayed by the detection method of the presentinvention may be obtained from any source, so long as it is a sampleoriginating in a human being and containing the genomic DNA. The genomicDNA can be extracted and purified from such a sample in accordance withthe conventional method.

From this genomic DNA, a DNA region containing the mutation siterelating to the present invention is amplified. Thus a concentratedspecimen can be obtained in a large amount. For example, PCR can beperformed with the use of appropriate primers by which a regioncontaining the above-mentioned mutation in the exon 13 or 22 alone canbe specifically amplified. These primers can be selected in theconventional manner. The base length of the region to be amplified isnot particularly restricted but usually regulated to 100 to 500 bp. Itis proper to employ, for example, primers homologous to the flankingintronic sequence (Seino, S., Seino, M. and Bell, Gl., Diabetes,39:123-128, 1990) which were employed in Examples given hereinafter inorder to amplify a region including the exon 13. To amplify a regionincluding the exon 22, use can be made of, for example, a sense primer(5'-CACTGACCTCATGCGCATGTGCTGG-3') (SEQ ID NO:3) and an antisense primer(5'-ATTGGACCGAGGCAAGGTCAGAAT-3') (SEQ ID NO:4) are employed. By usingthese primers, the desired regions as described above can be obtained asamplified DNA fragments of 322 bp (exon 13) and 346 bp (exon 22)respectively.

By using the desired DNA region which has been amplified by PCR, thespecific mutation of the present invention contained in this region canbe detected and confirmed. In the Examples given hereinafter, themutation in the exon 13 was detected by the RFLP method. The mutation atAla⁸³¹ (GCG) results in the specific cleavage site of the restrictionenzyme CfoI. When the above-mentioned PCR amplification product of theexon 13 (322 bp) having this mutation is digested with CfoI, therefore,two fragments (102 bp, 220 bp) are obtained. In contrast, no suchcleavage occurs in the mutation-free wild type (322 bp). The fragmentsthus formed are identified as specific bands by the conventional method.

To detect the mutation in the exon 22, use is made of allele-specifichybridization wherein the above-mentioned DNA fragment for diagnosticprobe is employed. This hybridization can be performed in a conventionalmanner, so long as it aims at detecting the mutation as specified in thepresent invention with the use of the specific diagnostic probe asdescribed above. For example, the following conditions were employed inExamples given hereinafter.

The PCR amplification product (346 bp) of the exon 22 as describedabove, which had been transferred onto a nitrocellulose filter, washybridized overnight at 30° C. in a probe solution containing 6× SSC,10× Denhardt's solution, 1% of SDS, 1 mg/ml of salmon sperm DNA and a ³²P-labeled probe. The hybridized filter was washed twice with 6× SSC in0.1% of SDS at 54° C. each for 20 minutes and then autoradio-graphed.The probe specific to the IR^(C1334) mutation (Cys¹³³⁴ :TGC), by whichit is distinguishable from the wild type (Tyr¹³³⁴ :TAC), employed hereinwas the ³² P-labeled probe 5'-ATGTGTGTGCAAGGGATGT-3' (SEQ ID NO:5).

The occurrence of the mutation of the present invention can be detectedby observing the pattern of the bands thus obtained (two hybridizedbands of 102 bp and 220 bp assignable to the mutation in the exon 13, orone of 346 bp assignable to the mutation in the exon 22) and confirmingthe same.

In this gene diagnosis, it is advantageous to utilize a diagnostic drugwhich contains as the active ingredient a means or reagent for detectingthe occurrence of the mutation according to the present invention.Therefore, the present invention further provides such a drug for thediagnosis of non-insulin-dependent diabetes mellitus. This diagnosticdrug contains as the essential ingredient a specific reagent suitablefor the method for detecting the occurrence of the mutation of thepresent invention. This specific reagent may be appropriately selecteddepending on the employed detection method. It is characterized by beingnecessary in the means for specifically detecting the mutation of thepresent invention, for example, the DNA fragment for diagnostic probe asdescribed above and/or a specific restriction enzyme. Although a reagentfor PCR-amplifying specifically a region of the mutation of the presentinvention (for example, a primer designed therefor, etc.) cannot beregarded as the essential ingredient of the diagnostic drug of thepresent invention, the diagnostic drug of the present invention maycontain such a reagent together with the reagent(s) for hybridization.

To further illustrate the present invention in greater detail, and notby way of limitation, the following Examples will be given.

EXAMPLES Example 1

Separation of human chromosomal DNA

Ten ml portions of blood of about 100 typical Japanese patients withNIDDM were provided by Dr. Makino et al. of the Second Department ofInternal Medicine, Faculty of Medicine, Chiba University. Humanchromosomal DNA was separated in the following manner.

1) Into two 50 ml blue-capped tubes were introduced 45 ml portions ofsolution A 0.32 M of sucrose, 10 mM of Tris-HCl (pH 7.5), 5 mM of MgCl₂and 1% of Triton X-100! with a 10 ml graduated pipet.

2) The blood was collected in an amount of about 10 ml.

3) About 5 ml portions of the blood were added to the two blue-cappedtubes containing the solution A followed by end-over-end mixing.

4) Each mixture was centrifuged at 4° C. for 10 minutes at 3,000 rpm.

5) The supernatant was carefully discarded and the tube was placedupside-down on Kim-Wipe to thereby eliminate the solution.

6) Into this tube was introduced 4 ml of solution B 0.075 M of NaCl,0.024 M of EDTA (pH 8.0)! with a 5 ml graduated pipet. After mixing,pellets were peeled off from the bottom of the tube, transferred intoanother tube and mixed well with a vortex mixer.

7) To this mixture was added 1 ml of solution C (containing equivalentamounts of 5% of SDS and 2 mg/ml of proteinase K) with an automaticpipet. After mixing well, the mixture was reacted overnight at 37° C.

8) Five ml of a phenol solution was added thereto with a 5 ml graduatedpipet and, after capping, mixed well. Further, 5 ml of a mixture ofchloroform with isoamyl alcohol (24:1) was added thereto and, aftercapping, mixed well.

9) The whole solution was transferred into an orange-capped conical tube(15 ml) and centrifuged at 3,000 rpm for 10 minutes.

10) The supernatant was taken up carefully with a Pasteur pipet having acut-off tip and transferred into a fresh orange-capped tube. Then 5 mlof a mixture of phenol with chloroform (1:1) was added thereto and mixedfor 30 minutes.

11) The mixture was centrifuged at 3,000 rpm for 10 minutes.

12) The supernatant was taken up carefully with a Pasteur pipet having acut-off tip and transferred into a blue-capped tube (50 ml).

13) After adding 0.5 ml of solution D (3 M sodium acetate) with anautomatic pipet, capping and mixing, 10 ml of cold ethanol (99.9%) wasslowly layered thereon with a graduated pipet.

14) After capping, the mixture was slowly mixed end-over-end. Thus, thechromosomal DNA was obtained as a white insoluble matter. It was scoopedup with a Pasteur pipet having a bowed tip and gently immersed in 1 mlof a 70% ethanol solution for about 15 seconds. Next, the insolublematter was transferred into an Eppendorf tube containing 200 μl ofsolution E 10 mM of Tris-HCl (pH 7.5), 1 mM of EDTA).

15) The Eppendorf tube was capped and the contents were mixed with avortex mixer several times to thereby well dissolve the DNA.

Similarly, chromosomal DNA was separated from 272 healthy volunteers.

Example 2

Amplification of insulin receptor gene exons by PCR, subcloning thereofand isolation of plasmid DNA

In accordance with the method of Seino et al. (Proc. Natl. Acad. Sci.USA 86:114-118, 1989; Diabetes 39:123-128, 1990), the insulin receptorgene was amplified by PCR with the use of primer DNAs by which all of 22exons of the insulin receptor gene could be amplified.

Namely, 99 μl of mixed solution F of the following composition wasintroduced into a 0.5 ml tube. Next, 1 μl (1 μg) of the DNA was addedthereto and mixed therewith.

    ______________________________________    (Volume)           (Final)    ______________________________________    10 x reaction buffer                       10.0 μl                               1 x    dNTPs mix (2.5 mM) 8.0 μl                               200 μM    upstream primer    0.5 μl                               50 pmol/100 μl    downstream primer  0.5 μl                               50 pmol/100 μl    H.sub.2 O          79.5 μl    Ampli Taq ™ polymerase                       0.5 μl                               2.5 U/assay    total              99.0 μl.    ______________________________________

Mineral oil was layered onto the above mixture so as to prevent thesample from evaporating. To improve the heat conductivity, a drop ofmineral oil was further added to each well of the heat box. By usingthem, PCR was performed under the conditions as specified below.

    ______________________________________    1. Initial denaturation                     94° C., 3 minutes.    2. Denaturation  94° C., 1 minute.    3. Annealing     53° C., 1.5 minutes × 30 cycles.    4. Extension     72° C., 2.5 minutes,                     terminated at 72° C., 4 minutes.    ______________________________________

To the sample thus amplified by PCR was added 100 μl of chloroform.After mixing well with a vortex mixer, the mixture was centrifuged for 1minute. Then the lower layer was thoroughly eliminated with a Pasteurpipet and DNA was obtained from the upper layer.

The PCR amplified DNA fragments thus obtained were purified by agarosegel electrophoresis and ligated to the HincII site of pUC19 which hadbeen treated with alkaline phosphatase. Then these recombinants wereintroduced into Escherichia coli. From several hundred colonies tolerantto ampicillin, 12 colonies were picked up. Then it was thus confirmedfor each exon that more than 50% of the colonies had a PCR DNA fragment.All of the remaining colonies were scratched off from the agar mediumand grown in a liquid medium. Then crude plasmid DNAs were separated bythe alkaline lysis method and RNAs were removed by precipitation withpolyethylene glycol. It is considered that, when a number of E. colitransformants are used as in the case of the present Example, thepatroclinal PCR products and the matroclinal ones are obtained almost inthe same amount. Accordingly, the base sequences of the DNAs thusseparated have both of the patroclinal and matroclinal gene sequencestherein.

Example 3

DNA sequencing

The DNA fragments obtained in Example 2 were sequenced by the dideoxysequencing method with the use of in isotope (Maxam, A. M. and Gilbert,W., Proc. Natl. Acad. Sci. USA 74:560-564, 1977). In brief, the primersemployed in the DNA amplification by PCR were hybridized with the DNAfragments. Then DNAs were synthesized with Sequenase by usingisotope-labeled dCTP, followed by electrophoresis and autoradiography.

As a result, mutations free from any amino acid replacement were foundin ten patients while mutations accompanied by amino acid replacementwere found in four patients.

Among the four patients with mutations accompanied by amino acidreplacement as described above, three patients showed the mutation(Thr⁸³¹ →Ala⁸³¹) in the exon 13 of the insulin receptor β-subunit, asshown below.

    ______________________________________    Val.sup.830 - Thr.sup.831 - His.sup.832    GTGmal        5'    ACG    CAT    3'    CAC           3'    TGC    GTA    5'    GTGant        5'    GCG    CAT    3'    CAC           3'    CGC    GTA    5'    Val.sup.830 - Ala.sup.831 - His.sup.832.    ______________________________________

FIG. 1 is an electrophorogram which shows a part of the DNA basesequence of the exon 13 of the insulin receptor β-subunit of fivepatients with NIDDM involving those having the above mutation. It wasfound that the patient of the third lane was a heterozygote having bothof the sequences Thr⁸³¹ (ACG) and Ala⁸³¹ (GCG). Two other patients hadthe same sequences.

On the other hand, the remaining patient having mutation accompaniedwith amino acid replacement showed the following mutation (Tyr¹³³⁴→Cys¹³³⁴) in the exon 22 of the insulin receptor β-subunit, as shownbelow.

    ______________________________________    Pro.sup.1333 - Tyr.sup.1334 - Thr.sup.1335    CCTmal        5'    TAC    ACA    3'    GGA           3'    ATG    TGT    5'    CCTant        5'    TGC    ACA    3'    GGA           3'    ACG    TGT    5'    Pro.sup.1333 - Cys.sup.1334 - Thr.sup.1335.    ______________________________________

FIG. 2 is an electrophorogram which shows a part of the DNA basesequence of the exon 22 of the insulin receptor β-subunit of eightpatients with NIDDM involving the one having the above mutation. It wasfound that the patient of the seventh lane was a heterozygote havingboth of the sequences Tyr¹³³⁴ (TAC) and Cys¹³³⁴ (TGC).

Example 4

Functional characterization of mutant insulin receptors expressed inmammalian cells

Test Method

(1) Construction of expression plasmids

By using PCR on cDNA, two artificially mutated cDNAs, i.e., insulinreceptors IR^(A831) having the mutation (Thr⁸³¹ →Ala⁸³¹) and IR^(C1334)having the mutation (Tyr¹³³⁴ →Cys¹³³⁴) were constructed.

Next, the artificial mutant cDNAs IR^(A831) and IR^(C1334) weresubcloned into a mammalian expression vector SRα (Y. Tanabe et al., Mol.Cell Biol. 8:446-472, 1988) to thereby give SRαIR^(A831) andSRαIR^(C1334) respectively. The wild type insulin receptor SRαIR^(WT)employed as the control was constructed by a method of F. Kanai et al.(J. Bio. Chem. 268:14523-14526, 1993).

(2) Establishment of CHO cells expressing the wild type and artificialmutant insulin receptors

CHO cells were transfected with SRαIR^(WT), SRαIR^(A831) orSRαIR^(C1334) (each 10 μg) and pSV2-neo (1 μg). After selecting with 400μg/ml of G418 (Sigma), cells expressing human insulin receptors wereidentified by ¹²⁵ I-labeled insulin binding in accordance with themethod of H. Hayashi et al. (Biochem. J. 280:769-775, 1991). The numberof cell surface receptors was calculated by Scatchard analysis (G.Scathard, Ann. NY Acad. Sci. 51:660-672, 1949).

(3) Assay of receptor tyrosine kinase activities

By using the method of Hayashi et al. (Biochem. J. 280:769-775, 1991),the insulin-stimulated receptor autophosphorylation of IR^(WT) andIR^(A831) was performed in a 96-well plate. The incorporation of ³² Pinto the receptor β-subunit was detected by 6% SDS-PAGE and measured bya Bio-image-analyzer BAS2000.

(4) Insulin-induced complex formation of insulin receptors and α-typep85 subunit of PI 3-kinase.

To transiently express both of the α-type p85 subunit of PI 3-kinase andwild type or mutant IRs, COS-7 cells were transfected with SRαp85α (1.5μg) and SRαIR, SRαIR^(A831) or SRαIR^(C1334) (each 1.5 μg), respectivelyby using Lipofectamine™ Reagent (Bethesda Research Laboratories). Afterstimulating with 10⁻⁷ M of insulin for 10 minutes, cell lysates wereprepared and incubated with an anti-IR antibody 1G2, which recognizesthe β-subunit of IR, or a rabbit polyclonal anti-p85α antibody, andprotein G-Sepharose. The immunoprecipitates were electrophoresed on a 6%SDS-PAGE. Immunoblotting was performed by using an anti-phosphotyrosineantibody (PY20) or an anti-insulin receptor antibody 3B11 whichrecognizes the α-subunit of IR.

Results

(1) A831 mutation

Examination was made on two clones stably expressing IR^(WT) (clone Nos.12 and 21) and two clones stably expressing IR^(A831) (clone Nos. 10 and17) obtained in the above test method (2).

FIG. 3A shows the Scatchard analysis of IR^(WT) (clone Nos. 12 and 21)and IR^(A831) (clone Nos. 10 and 17). The number of insulin bindingsites in each clone were counted. As a result, the number of highaffinity binding sites were 1.1 and a.5×10⁶ per cell in IR^(WT) and 0.5and 0.9×10⁹ per cell in IR^(A831), and the dissociation constants (Kd)were 2.8 and 3.5 nM for IR^(WT) and 1.7 and 2.7 nM for IR^(A831).IR^(WT) and IR^(A831) showed each the occurrence of dissociation with adecrease in pH (from 7.5 to 5.5) and no difference was observed betweenthe wild type and the mutant IRs.

Next, the autophosphorylation activities of these receptors weredetermined by the test method (3) described above. FIG. 3B shows theresults thus obtained. As FIG. 3B clearly shows, the activities ofreceptor autophosphorylation were increased in association with thereceptor numbers. Half-maximal stimulation occurred at 5.5 and 5.4×10⁻¹⁰M for IR^(WT) and at 5.0 and 4.9×10⁻¹⁰ for IR^(A831), showing noremarkable difference between the wild type and the mutant IRs.

Further, the tyrosine kinase activity toward an endogenous substrateIRS-1 was determined by immunoblotting with the use of ananti-phosphotyrosine antibody (PY-20). FIG. 3C shows the results. AsFIG. 3C clearly shows, the IRS-1 (molecular weight: 160,000) wasphosphorylated insulin-dependently at tyrosine residues in parallel tothe autophosphorylation rate of the receptor β-subunit (molecularweight: 95,000).

Subsequently, the insulin binding and receptor autophosphorylation wereexamined by using COS cells transiently expressing IR^(WT) and IR^(A831)so as to avoid differences among CHO clones stably expressing each IR.No remarkable difference was observed between the wild type andartificial mutant receptors in either insulin binding affinity or inreceptor kinase activity. Similarly, no distinct difference was observedbetween the wild type and artificial mutant receptors in receptorprocessing, internalization, degradation, insulin-stimulated glucoseuptake, glycogen synthesis and DNA synthesis. That is to say, no directevidence was found regarding the receptor function disorders in the A831mutation.

(2) C1334 mutation

The above-mentioned experiments performed on IR^(A831) were repeated onIR^(C1334) to thereby examine the receptor function disorders inIR^(C1334).

No distinct difference in the functions between IR^(C1334) and RI^(WT)was found out in the examination with the use of CHO clones stablyexpressing IRs.

However, a difference was observed in the affinity to PI 3-kinase. It isknown that the autophosphorylated Tyr¹³³⁴ of IR locates in the putativebinding motif Y(P)XXM! to the SH2 domains of the 85-kDa regulatorysubunit (p85) of phosphatidylinositol 3-kinase (D. J. Van Horn et al.,J. Biol. Chem. 269:29-32, 1994). Since the binding of autophosphorylatedIR to PI 3-kinase in response to insulin leads to the activation of thisenzyme, this mechanism might be an alternative pathway for theactivation of PI 3-kinase by the binding of IRS-1. The present inventorshad formerly reported that PI 3-kinase mediates the translocation ofglucose transporter type 4 (GLUT4) (see, for example, F. Kanai et al.,Biochem. Biophys. Res. Commun. 195:762-768, 1993). Thus examination wasmade on the direct interactions between IR^(C1334) and p85 of PI3-kinase.

First, the p85 subunit of PI 3-kinase was transiently expressed in COScells with the use of IR^(WT), IR^(A831) and IR^(C1334). After treatingwith insulin, the cell lysates were precipitated by either an anti-IRβantibody (αIRβ) or an anti-p85 antibody (αp85) (FIG. 4). Theimmunoprecipitates were examined by immunoblotting with ananti-phosphotyrosine antibody (αPY) (FIG. 4A) or an anti-IR α-subunitantibody (αIRα) (FIG. 4B). In the transient expression system, theinsulin treatment stimulated the tyrosine phosphorylation of the p85subunit of PI 3-kinase and the binding of the p85 to IR^(WT). Whenimmunoblotted with anti-IRα antibody, the anti-IRβ antibody precipitatedIR^(WT),IR A831 and IR^(C1334) to almost the same degree (FIG. 4B). Whenimmunoblotted with anti-p85 antibody, the anti-p85 antibody precipitatedIR^(WT), IR^(A831) and IR^(C1334) to almost the same degree.

Subsequently, examination was made on the tyrosine phosphorylation ofp85 and IRβ, and the binding of p85 to IR^(WT), IR^(A831) andIR^(C1334). When immunoblotted with anti-phosphotyrosine antibody (FIG.4A), all of the IRs phosphorylated p85 at tyrosine residues to the sameextent. The tyrosine phosphorylation of IR^(C1334) was reduced comparedwith IR^(WT) and IR^(A831) (FIG. 4A). This reduction did not relate tothe decrease in the tyrosine kinase activity of IR^(C1334). In theexamination on the incorporation of ³² P into the substrates, IR^(C1334)showed almost the same activity toward the autophosphorylation sites andan exogenous substrate poly(Glu, Tyr) 4:1 as IR^(WT) and IR^(A831) inCHO cells expressing these IRs. Thus it is considered that theantiphosphotyrosine antibody would preferably recognize the site ofautophosphorylated Y¹³³⁴ more than other autophosphorylation sites ofIRβ. The anti-p85 antibody co-precipitated with IR^(WT) and IR^(A831)but not with IR^(C1334) (FIG. 4B). This fact means that IR^(C1334) wouldnot bind to p85 and that the major binding site of IR to p85 might bethe Y¹³³⁴ residue.

Example 5

Statistical Processing

272 healthy volunteers were analyzed to find those having the mutantinsulin receptor DNA (Thr⁸³¹ →Ala⁸³¹). As a result, none of thevolunteers showed the mutation. Thus, the relation between the mutantinsulin receptor DNA of the present invention and NIDDM was testedthrough statistical processing by chi-square analysis. The results aregiven in the following Table 1.

                  TABLE 1    ______________________________________    Analysis on the frequency of the occurrence of insulin    receptor β-subunit mutation (T.sup.831 → A.sup.831)    in NIDDM and non-diabetic subjects    NIDDM            Non-diabetic.sup.1)                                Total    ______________________________________    A.sup.831             3.sup.2)    0          3    T.sup.831            48           272        320    Total   51           272        323    ______________________________________     .sup.1) The nondiabetic subjects were not examined by OGTT, etc. but     selected by inquiring whether they had any medical history or family     histories of diabetes in their immediate parents or grandparents.     .sup.2) The mutation (Thr.sup.831 → Ala.sup.831) significantly     related to the onset of NIDDM in the chisquare analysis with Yates     correction (p < 0.05). Supposing that the spontaneous onset rate of NIDDM     is 5% and 5% (14 subjects) of the 272 nondiabetic subjects might migrate     into the NIDDM T.sup.831 group, the mutation still shows a significant     difference in the above calibration.

Example 6

Pedigree-linkage Analysis

(1) Relationship between IR^(C133) and the onset of NIDDM

To examine the relationship between IR^(C1334) and the onset of NIDDM,data on the family with IR^(C1334) were analyzed.

In this family, the mother and the second son were reported to bediabetics, and the other two children (T. S. and S. K.) in addition tothe proband (Y. Y.) were diagnosed as diabetics (FIG. 5A). When examinedby the allele specific hybridization at TAC (Tyr¹³³⁴)→TGC (Cys¹³³⁴)(FIG. 5B), however, two of them (T. S. and S. K.) had the normal IR(IR^(Y1334)) while the proband alone had IR^(C1334). Thus the results ofthis pedigree-linkage analysis indicate that the mutant IR^(C1334) isnot the common cause of the onset of NIDDM in this family.

(2) Relationship between IR^(A831) and the onset of NIDDM

To examine the relationship between IR^(A831) and the onset of NIDDM,data on the family with IR^(A831) were analyzed. The deceased proband'sfather had NIDDM, but his mutation could not be confirmed. The mothercould not be subjected to the examination due to her advanced age. Allfour siblings were heterozygotes of IR^(A831), and three had NIDDM whileone had an impaired glucose tolerance (IGT), i.e., intermediate betweennormal and diabetes (FIG. 6). IR^(A831) was detected by theabove-mentioned method with the formation of the specific cleavage siteof the restriction enzyme CfoI (FIG. 6B). Consanguineous marriage in thefamily was ruled out. The allele of IR^(A831) may have been derived fromthe father. This pedigree and linkage analysis data strongly suggestthat the heterozygous mutation IR^(A831) would be responsible for theonset of NIDDM.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 5    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 4149 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..4146    -     (ix) FEATURE:              (A) NAME/KEY: mat.sub.-- - #peptide              (B) LOCATION: 82..4146    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - ATG GGC ACC GGG GGC CGG CGG GGG GCG GCG GC - #C GCG CCG CTG CTG GTG      48    Met Gly Thr Gly Gly Arg Arg Gly Ala Ala Al - #a Ala Pro Leu Leu Val    15    - GCG GTG GCC GCG CTG CTA CTG GGC GCC GCG GG - #C CAC CTG TAC CCC GGA      96    Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gl - #y His Leu Tyr Pro Gly    #  5  1    - GAG GTG TGT CCC GGC ATG GAT ATC CGG AAC AA - #C CTC ACT AGG TTG CAT     144    Glu Val Cys Pro Gly Met Asp Ile Arg Asn As - #n Leu Thr Arg Leu His    #                 20    - GAG CTG GAG AAT TGC TCT GTC ATC GAA GGA CA - #C TTG CAG ATA CTC TTG     192    Glu Leu Glu Asn Cys Ser Val Ile Glu Gly Hi - #s Leu Gln Ile Leu Leu    #             35    - ATG TTC AAA ACG AGG CCC GAA GAC TTC CGA GA - #C CTC AGT TTC CCC AAA     240    Met Phe Lys Thr Arg Pro Glu Asp Phe Arg As - #p Leu Ser Phe Pro Lys    #         50    - CTC ATC ATG ATC ACT GAT TAC TTG CTG CTC TT - #C CGG GTC TAT GGG CTC     288    Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Ph - #e Arg Val Tyr Gly Leu    #     65    - GAG AGC CTG AAG GAC CTG TTC CCC AAC CTC AC - #G GTC ATC CGG GGA TCA     336    Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Th - #r Val Ile Arg Gly Ser    # 85    - CGA CTG TTC TTT AAC TAC GCG CTG GTC ATC TT - #C GAG ATG GTT CAC CTC     384    Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Ph - #e Glu Met Val His Leu    #                100    - AAG GAA CTC GGC CTC TAC AAC CTG ATG AAC AT - #C ACC CGG GGT TCT GTC     432    Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Il - #e Thr Arg Gly Ser Val    #           115    - CGC ATC GAG AAG AAC AAT GAG CTC TGT TAC TT - #G GCC ACT ATC GAC TGG     480    Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Le - #u Ala Thr Ile Asp Trp    #       130    - TCC CGT ATC CTG GAT TCC GTG GAG GAT AAT CA - #C ATC GTG TTG AAC AAA     528    Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Hi - #s Ile Val Leu Asn Lys    #   145    - GAT GAC AAC GAG GAG TGT GGA GAC ATC TGT CC - #G GGT ACC GCG AAG GGC     576    Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pr - #o Gly Thr Ala Lys Gly    150                 1 - #55                 1 - #60                 1 -    #65    - AAG ACC AAC TGC CCC GCC ACC GTC ATC AAC GG - #G CAG TTT GTC GAA CGA     624    Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gl - #y Gln Phe Val Glu Arg    #               180    - TGT TGG ACT CAT AGT CAC TGC CAG AAA GTT TG - #C CCG ACC ATC TGT AAG     672    Cys Trp Thr His Ser His Cys Gln Lys Val Cy - #s Pro Thr Ile Cys Lys    #           195    - TCA CAC GGC TGC ACC GCC GAA GGC CTC TGT TG - #C CAC AGC GAG TGC CTG     720    Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cy - #s His Ser Glu Cys Leu    #       210    - GGC AAC TGT TCT CAG CCC GAC GAC CCC ACC AA - #G TGC GTG GCC TGC CGC     768    Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Ly - #s Cys Val Ala Cys Arg    #   225    - AAC TTC TAC CTG GAC GGC AGG TGT GTG GAG AC - #C TGC CCG CCC CCG TAC     816    Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Th - #r Cys Pro Pro Pro Tyr    230                 2 - #35                 2 - #40                 2 -    #45    - TAC CAC TTC CAG GAC TGG CGC TGT GTG AAC TT - #C AGC TTC TGC CAG GAC     864    Tyr His Phe Gln Asp Trp Arg Cys Val Asn Ph - #e Ser Phe Cys Gln Asp    #               260    - CTG CAC CAC AAA TGC AAG AAC TCG CGG AGG CA - #G GGC TGC CAC CAA TAC     912    Leu His His Lys Cys Lys Asn Ser Arg Arg Gl - #n Gly Cys His Gln Tyr    #           275    - GTC ATT CAC AAC AAC AAG TGC ATC CCT GAG TG - #T CCC TCC GGG TAC ACG     960    Val Ile His Asn Asn Lys Cys Ile Pro Glu Cy - #s Pro Ser Gly Tyr Thr    #       290    - ATG AAT TCC AGC AAC TTG CTG TGC ACC CCA TG - #C CTG GGT CCC TGT CCC    1008    Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cy - #s Leu Gly Pro Cys Pro    #   305    - AAG GTG TGC CAC CTC CTA GAA GGC GAG AAG AC - #C ATC GAC TCG GTG ACG    1056    Lys Val Cys His Leu Leu Glu Gly Glu Lys Th - #r Ile Asp Ser Val Thr    310                 3 - #15                 3 - #20                 3 -    #25    - TCT GCC CAG GAG CTC CGA GGA TGC ACC GTC AT - #C AAC GGG AGT CTG ATC    1104    Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Il - #e Asn Gly Ser Leu Ile    #               340    - ATC AAC ATT CGA GGA GGC AAC AAT CTG GCA GC - #T GAG CTA GAA GCC AAC    1152    Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Al - #a Glu Leu Glu Ala Asn    #           355    - CTC GGC CTC ATT GAA GAA ATT TCA GGG TAT CT - #A AAA ATC CGC CGA TCC    1200    Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Le - #u Lys Ile Arg Arg Ser    #       370    - TAC GCT CTG GTG TCA CTT TCC TTC TTC CGG AA - #G TTA CGT CTG ATT CGA    1248    Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Ly - #s Leu Arg Leu Ile Arg    #   385    - GGA GAG ACC TTG GAA ATT GGG AAC TAC TCC TT - #C TAT GCC TTG GAC AAC    1296    Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Ph - #e Tyr Ala Leu Asp Asn    390                 3 - #95                 4 - #00                 4 -    #05    - CAG AAC CTA AGG CAG CTC TGG GAC TGG AGC AA - #A CAC AAC CTC ACC ACC    1344    Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Ly - #s His Asn Leu Thr Thr    #               420    - ACT CAG GGG AAA CTC TTC TTC CAC TAT AAC CC - #C AAA CTC TGC TTG TCA    1392    Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pr - #o Lys Leu Cys Leu Ser    #           435    - GAA ATC CAC AAG ATG GAA GAA GTT TCA GGA AC - #C AAG GGG CGC CAG GAG    1440    Glu Ile His Lys Met Glu Glu Val Ser Gly Th - #r Lys Gly Arg Gln Glu    #       450    - AGA AAC GAC ATT GCC CTG AAG ACC AAT GGG GA - #C AAG GCA TCC TGT GAA    1488    Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly As - #p Lys Ala Ser Cys Glu    #   465    - AAT GAG TTA CTT AAA TTT TCT TAC ATT CGG AC - #A TCT TTT GAC AAG ATC    1536    Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Th - #r Ser Phe Asp Lys Ile    470                 4 - #75                 4 - #80                 4 -    #85    - TTG CTG AGA TGG GAG CCG TAC TGG CCC CCC GA - #C TTC CGA GAC CTC TTG    1584    Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro As - #p Phe Arg Asp Leu Leu    #               500    - GGG TTC ATG CTG TTC TAC AAA GAG GCC CCT TA - #T CAG AAT GTG ACG GAG    1632    Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Ty - #r Gln Asn Val Thr Glu    #           515    - TTC GAT GGG CAG GAT GCG TGT GGT TCC AAC AG - #T TGG ACG GTG GTA GAC    1680    Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Se - #r Trp Thr Val Val Asp    #       530    - ATT GAC CCA CCC CTG AGG TCC AAC GAC CCC AA - #A TCA CAG AAC CAC CCA    1728    Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Ly - #s Ser Gln Asn His Pro    #   545    - GGG TGG CTG ATG CGG GGT CTC AAG CCC TGG AC - #C CAG TAT GCC ATC TTT    1776    Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Th - #r Gln Tyr Ala Ile Phe    550                 5 - #55                 5 - #60                 5 -    #65    - GTG AAG ACC CTG GTC ACC TTT TCG GAT GAA CG - #C CGG ACC TAT GGG GCC    1824    Val Lys Thr Leu Val Thr Phe Ser Asp Glu Ar - #g Arg Thr Tyr Gly Ala    #               580    - AAG AGT GAC ATC ATT TAT GTC CAG ACA GAT GC - #C ACC AAC CCC TCT GTG    1872    Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Al - #a Thr Asn Pro Ser Val    #           595    - CCC CTG GAT CCA ATC TCA GTG TCT AAC TCA TC - #A TCC CAG ATT ATT CTG    1920    Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Se - #r Ser Gln Ile Ile Leu    #       610    - AAG TGG AAA CCA CCC TCC GAC CCC AAT GGC AA - #C ATC ACC CAC TAC CTG    1968    Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly As - #n Ile Thr His Tyr Leu    #   625    - GTT TTC TGG GAG AGG CAG GCG GAA GAC AGT GA - #G CTG TTC GAG CTG GAT    2016    Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Gl - #u Leu Phe Glu Leu Asp    630                 6 - #35                 6 - #40                 6 -    #45    - TAT TGC CTC AAA GGG CTG AAG CTG CCC TCG AG - #G ACC TGG TCT CCA CCA    2064    Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Ar - #g Thr Trp Ser Pro Pro    #               660    - TTC GAG TCT GAA GAT TCT CAG AAG CAC AAC CA - #G AGT GAG TAT GAG GAT    2112    Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gl - #n Ser Glu Tyr Glu Asp    #           675    - TCG GCC GGC GAA TGC TGC TCC TGT CCA AAG AC - #A GAC TCT CAG ATC CTG    2160    Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Th - #r Asp Ser Gln Ile Leu    #       690    - AAG GAG CTG GAG GAG TCC TCG TTT AGG AAG AC - #G TTT GAG GAT TAC CTG    2208    Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Th - #r Phe Glu Asp Tyr Leu    #   705    - CAC AAC GTG GTT TTC GTC CCC AGA AAA ACC TC - #T TCA GGC ACT GGT GCC    2256    His Asn Val Val Phe Val Pro Arg Lys Thr Se - #r Ser Gly Thr Gly Ala    710                 7 - #15                 7 - #20                 7 -    #25    - GAG GAC CCT AGG CCA TCT CGG AAA CGC AGG TC - #C CTT GGC GAT GTT GGG    2304    Glu Asp Pro Arg Pro Ser Arg Lys Arg Arg Se - #r Leu Gly Asp Val Gly    #               740    - AAT GTG ACG GTG GCC GTG CCC ACG GTG GCA GC - #T TTC CCC AAC ACT TCC    2352    Asn Val Thr Val Ala Val Pro Thr Val Ala Al - #a Phe Pro Asn Thr Ser    #           755    - TCG ACC AGC GTG CCC ACG AGT CCG GAG GAG CA - #C AGG CCT TTT GAG AAG    2400    Ser Thr Ser Val Pro Thr Ser Pro Glu Glu Hi - #s Arg Pro Phe Glu Lys    #       770    - GTG GTG AAC AAG GAG TCG CTG GTC ATC TCC GG - #C TTG CGA CAC TTC ACG    2448    Val Val Asn Lys Glu Ser Leu Val Ile Ser Gl - #y Leu Arg His Phe Thr    #   785    - GGC TAT CGC ATC GAG CTG CAG GCT TGC AAC CA - #G GAC ACC CCT GAG GAA    2496    Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gl - #n Asp Thr Pro Glu Glu    790                 7 - #95                 8 - #00                 8 -    #05    - CGG TGC AGT GTG GCA GCC TAC GTC AGT GCG AG - #G ACC ATG CCT GAA GCC    2544    Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Ar - #g Thr Met Pro Glu Ala    #               820    - AAG GCT GAT GAC ATT GTT GGC CCT GTG ACG CA - #T GAA ATC TTT GAG AAC    2592    Lys Ala Asp Asp Ile Val Gly Pro Val Thr Hi - #s Glu Ile Phe Glu Asn    #           835    - AAC GTC GTC CAC TTG ATG TGG CAG GAG CCG AA - #G GAG CCC AAT GGT CTG    2640    Asn Val Val His Leu Met Trp Gln Glu Pro Ly - #s Glu Pro Asn Gly Leu    #       850    - ATC GTG CTG TAT GAA GTG AGT TAT CGG CGA TA - #T GGT GAT GAG GAG CTG    2688    Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Ty - #r Gly Asp Glu Glu Leu    #   865    - CAT CTC TGC GTC TCC CGC AAG CAC TTC GCT CT - #G GAA CGG GGC TGC AGG    2736    His Leu Cys Val Ser Arg Lys His Phe Ala Le - #u Glu Arg Gly Cys Arg    870                 8 - #75                 8 - #80                 8 -    #85    - CTG CGT GGG CTG TCA CCG GGG AAC TAC AGC GT - #G CGA ATC CGG GCC ACC    2784    Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Va - #l Arg Ile Arg Ala Thr    #               900    - TCC CTT GCG GGC AAC GGC TCT TGG ACG GAA CC - #C ACC TAT TTC TAC GTG    2832    Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pr - #o Thr Tyr Phe Tyr Val    #           915    - ACA GAC TAT TTA GAC GTC CCG TCA AAT ATT GC - #A AAA ATT ATC ATC GGC    2880    Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Al - #a Lys Ile Ile Ile Gly    #       930    - CCC CTC ATC TTT GTC TTT CTC TTC AGT GTT GT - #G ATT GGA AGT ATT TAT    2928    Pro Leu Ile Phe Val Phe Leu Phe Ser Val Va - #l Ile Gly Ser Ile Tyr    #   945    - CTA TTC CTG AGA AAG AGG CAG CCA GAT GGG CC - #G CTG GGA CCG CTT TAC    2976    Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pr - #o Leu Gly Pro Leu Tyr    950                 9 - #55                 9 - #60                 9 -    #65    - GCT TCT TCA AAC CCT GAG TAT CTC AGT GCC AG - #T GAT GTG TTT CCA TGC    3024    Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Se - #r Asp Val Phe Pro Cys    #               980    - TCT GTG TAC GTG CCG GAC GAG TGG GAG GTG TC - #T CGA GAG AAG ATC ACC    3072    Ser Val Tyr Val Pro Asp Glu Trp Glu Val Se - #r Arg Glu Lys Ile Thr    #           995    - CTC CTT CGA GAG CTG GGG CAG GGC TCC TTC GG - #C ATG GTG TAT GAG GGC    3120    Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gl - #y Met Val Tyr Glu Gly    #      10105    - AAT GCC AGG GAC ATC ATC AAG GGT GAG GCA GA - #G ACC CGC GTG GCG GTG    3168    Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala Gl - #u Thr Arg Val Ala Val    #  10250    - AAG ACG GTC AAC GAG TCA GCC AGT CTC CGA GA - #G CGG ATT GAG TTC CTC    3216    Lys Thr Val Asn Glu Ser Ala Ser Leu Arg Gl - #u Arg Ile Glu Phe Leu    #               10451035 - #                1040    - AAT GAG GCC TCG GTC ATG AAG GGC TTC ACC TG - #C CAT CAC GTG GTG CGC    3264    Asn Glu Ala Ser Val Met Lys Gly Phe Thr Cy - #s His His Val Val Arg    #              10605    - CTC CTG GGA GTG GTG TCC AAG GGC CAG CCC AC - #G CTG GTG GTG ATG GAG    3312    Leu Leu Gly Val Val Ser Lys Gly Gln Pro Th - #r Leu Val Val Met Glu    #          10750    - CTG ATG GCT CAC GGA GAC CTG AAG AGC TAC CT - #C CGT TCT CTG CGG CCA    3360    Leu Met Ala His Gly Asp Leu Lys Ser Tyr Le - #u Arg Ser Leu Arg Pro    #      10905    - GAG GCT GAG AAT AAT CCT GGC CGC CCT CCC CC - #T ACC CTT CAA GAG ATG    3408    Glu Ala Glu Asn Asn Pro Gly Arg Pro Pro Pr - #o Thr Leu Gln Glu Met    #  11050    - ATT CAG ATG GCG GCA GAG ATT GCT GAC GGG AT - #G GCC TAC CTG AAC GCC    3456    Ile Gln Met Ala Ala Glu Ile Ala Asp Gly Me - #t Ala Tyr Leu Asn Ala    #               11251115 - #                1120    - AAG AAG TTT GTG CAT CGG GAC CTG GCA GCG AG - #A AAC TGC ATG GTC GCC    3504    Lys Lys Phe Val His Arg Asp Leu Ala Ala Ar - #g Asn Cys Met Val Ala    #              11405    - CAT GAT TTT ACT GTC AAA ATT GGA GAC TTT GG - #A ATG ACC AGA GAC ATC    3552    His Asp Phe Thr Val Lys Ile Gly Asp Phe Gl - #y Met Thr Arg Asp Ile    #          11550    - TAT GAA ACG GAT TAC TAC CGG AAA GGG GGC AA - #G GGT CTG CTC CCT GTA    3600    Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Ly - #s Gly Leu Leu Pro Val    #      11705    - CGG TGG ATG GCA CCG GAG TCC CTG AAG GAT GG - #G GTC TTC ACC ACT TCT    3648    Arg Trp Met Ala Pro Glu Ser Leu Lys Asp Gl - #y Val Phe Thr Thr Ser    #  11850    - TCT GAC ATG TGG TCC TTT GGC GTG GTC CTT TG - #G GAA ATC ACC AGC TTG    3696    Ser Asp Met Trp Ser Phe Gly Val Val Leu Tr - #p Glu Ile Thr Ser Leu    #               12051195 - #                1200    - GCA GAA CAG CCT TAC CAA GGC CTG TCT AAT GA - #A CAG GTG TTG AAA TTT    3744    Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Gl - #u Gln Val Leu Lys Phe    #              12205    - GTC ATG GAT GGA GGG TAT CTG GAT CAA CCC GA - #C AAC TGT CCA GAG AGA    3792    Val Met Asp Gly Gly Tyr Leu Asp Gln Pro As - #p Asn Cys Pro Glu Arg    #          12350    - GTC ACT GAC CTC ATG CGC ATG TGC TGG CAA TT - #C AAC CCC AAG ATG AGG    3840    Val Thr Asp Leu Met Arg Met Cys Trp Gln Ph - #e Asn Pro Lys Met Arg    #      12505    - CCA ACC TTC CTG GAG ATT GTC AAC CTG CTC AA - #G GAC GAC CTG CAC CCC    3888    Pro Thr Phe Leu Glu Ile Val Asn Leu Leu Ly - #s Asp Asp Leu His Pro    #  12650    - AGC TTT CCA GAG GTG TCG TTC TTC CAC AGC GA - #G GAG AAC AAG GCT CCC    3936    Ser Phe Pro Glu Val Ser Phe Phe His Ser Gl - #u Glu Asn Lys Ala Pro    #               12851275 - #                1280    - GAG AGT GAG GAG CTG GAG ATG GAG TTT GAG GA - #C ATG GAG AAT GTG CCC    3984    Glu Ser Glu Glu Leu Glu Met Glu Phe Glu As - #p Met Glu Asn Val Pro    #              13005    - CTG GAC CGT TCC TCG CAC TGT CAG AGG GAG GA - #G GCG GGG GGC CGG GAT    4032    Leu Asp Arg Ser Ser His Cys Gln Arg Glu Gl - #u Ala Gly Gly Arg Asp    #          13150    - GGA GGG TCC TCG CTG GGT TTC AAG CGG AGC TA - #C GAG GAA CAC ATC CCT    4080    Gly Gly Ser Ser Leu Gly Phe Lys Arg Ser Ty - #r Glu Glu His Ile Pro    #      13305    - TAC ACA CAC ATG AAC GGA GGC AAG AAA AAC GG - #G CGG ATT CTG ACC TTG    4128    Tyr Thr His Met Asn Gly Gly Lys Lys Asn Gl - #y Arg Ile Leu Thr Leu    #  13450    #                4149 TCC TAA    Pro Arg Ser Asn Pro Ser    1350                1355    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 1382 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Gly Thr Gly Gly Arg Arg Gly Ala Ala Al - #a Ala Pro Leu Leu Val    15    - Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gl - #y His Leu Tyr Pro Gly    #  5  1    - Glu Val Cys Pro Gly Met Asp Ile Arg Asn As - #n Leu Thr Arg Leu His    #                 20    - Glu Leu Glu Asn Cys Ser Val Ile Glu Gly Hi - #s Leu Gln Ile Leu Leu    #             35    - Met Phe Lys Thr Arg Pro Glu Asp Phe Arg As - #p Leu Ser Phe Pro Lys    #         50    - Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Ph - #e Arg Val Tyr Gly Leu    #     65    - Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Th - #r Val Ile Arg Gly Ser    # 85    - Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Ph - #e Glu Met Val His Leu    #                100    - Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Il - #e Thr Arg Gly Ser Val    #           115    - Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Le - #u Ala Thr Ile Asp Trp    #       130    - Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Hi - #s Ile Val Leu Asn Lys    #   145    - Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pr - #o Gly Thr Ala Lys Gly    150                 1 - #55                 1 - #60                 1 -    #65    - Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gl - #y Gln Phe Val Glu Arg    #               180    - Cys Trp Thr His Ser His Cys Gln Lys Val Cy - #s Pro Thr Ile Cys Lys    #           195    - Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cy - #s His Ser Glu Cys Leu    #       210    - Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Ly - #s Cys Val Ala Cys Arg    #   225    - Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Th - #r Cys Pro Pro Pro Tyr    230                 2 - #35                 2 - #40                 2 -    #45    - Tyr His Phe Gln Asp Trp Arg Cys Val Asn Ph - #e Ser Phe Cys Gln Asp    #               260    - Leu His His Lys Cys Lys Asn Ser Arg Arg Gl - #n Gly Cys His Gln Tyr    #           275    - Val Ile His Asn Asn Lys Cys Ile Pro Glu Cy - #s Pro Ser Gly Tyr Thr    #       290    - Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cy - #s Leu Gly Pro Cys Pro    #   305    - Lys Val Cys His Leu Leu Glu Gly Glu Lys Th - #r Ile Asp Ser Val Thr    310                 3 - #15                 3 - #20                 3 -    #25    - Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Il - #e Asn Gly Ser Leu Ile    #               340    - Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Al - #a Glu Leu Glu Ala Asn    #           355    - Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Le - #u Lys Ile Arg Arg Ser    #       370    - Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Ly - #s Leu Arg Leu Ile Arg    #   385    - Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Ph - #e Tyr Ala Leu Asp Asn    390                 3 - #95                 4 - #00                 4 -    #05    - Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Ly - #s His Asn Leu Thr Thr    #               420    - Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pr - #o Lys Leu Cys Leu Ser    #           435    - Glu Ile His Lys Met Glu Glu Val Ser Gly Th - #r Lys Gly Arg Gln Glu    #       450    - Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly As - #p Lys Ala Ser Cys Glu    #   465    - Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Th - #r Ser Phe Asp Lys Ile    470                 4 - #75                 4 - #80                 4 -    #85    - Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro As - #p Phe Arg Asp Leu Leu    #               500    - Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Ty - #r Gln Asn Val Thr Glu    #           515    - Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Se - #r Trp Thr Val Val Asp    #       530    - Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Ly - #s Ser Gln Asn His Pro    #   545    - Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Th - #r Gln Tyr Ala Ile Phe    550                 5 - #55                 5 - #60                 5 -    #65    - Val Lys Thr Leu Val Thr Phe Ser Asp Glu Ar - #g Arg Thr Tyr Gly Ala    #               580    - Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Al - #a Thr Asn Pro Ser Val    #           595    - Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Se - #r Ser Gln Ile Ile Leu    #       610    - Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly As - #n Ile Thr His Tyr Leu    #   625    - Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Gl - #u Leu Phe Glu Leu Asp    630                 6 - #35                 6 - #40                 6 -    #45    - Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Ar - #g Thr Trp Ser Pro Pro    #               660    - Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gl - #n Ser Glu Tyr Glu Asp    #           675    - Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Th - #r Asp Ser Gln Ile Leu    #       690    - Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Th - #r Phe Glu Asp Tyr Leu    #   705    - His Asn Val Val Phe Val Pro Arg Lys Thr Se - #r Ser Gly Thr Gly Ala    710                 7 - #15                 7 - #20                 7 -    #25    - Glu Asp Pro Arg Pro Ser Arg Lys Arg Arg Se - #r Leu Gly Asp Val Gly    #               740    - Asn Val Thr Val Ala Val Pro Thr Val Ala Al - #a Phe Pro Asn Thr Ser    #           755    - Ser Thr Ser Val Pro Thr Ser Pro Glu Glu Hi - #s Arg Pro Phe Glu Lys    #       770    - Val Val Asn Lys Glu Ser Leu Val Ile Ser Gl - #y Leu Arg His Phe Thr    #   785    - Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gl - #n Asp Thr Pro Glu Glu    790                 7 - #95                 8 - #00                 8 -    #05    - Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Ar - #g Thr Met Pro Glu Ala    #               820    - Lys Ala Asp Asp Ile Val Gly Pro Val Thr Hi - #s Glu Ile Phe Glu Asn    #           835    - Asn Val Val His Leu Met Trp Gln Glu Pro Ly - #s Glu Pro Asn Gly Leu    #       850    - Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Ty - #r Gly Asp Glu Glu Leu    #   865    - His Leu Cys Val Ser Arg Lys His Phe Ala Le - #u Glu Arg Gly Cys Arg    870                 8 - #75                 8 - #80                 8 -    #85    - Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Va - #l Arg Ile Arg Ala Thr    #               900    - Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pr - #o Thr Tyr Phe Tyr Val    #           915    - Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Al - #a Lys Ile Ile Ile Gly    #       930    - Pro Leu Ile Phe Val Phe Leu Phe Ser Val Va - #l Ile Gly Ser Ile Tyr    #   945    - Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pr - #o Leu Gly Pro Leu Tyr    950                 9 - #55                 9 - #60                 9 -    #65    - Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Se - #r Asp Val Phe Pro Cys    #               980    - Ser Val Tyr Val Pro Asp Glu Trp Glu Val Se - #r Arg Glu Lys Ile Thr    #           995    - Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gl - #y Met Val Tyr Glu Gly    #      10105    - Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala Gl - #u Thr Arg Val Ala Val    #  10250    - Lys Thr Val Asn Glu Ser Ala Ser Leu Arg Gl - #u Arg Ile Glu Phe Leu    #               10451035 - #                1040    - Asn Glu Ala Ser Val Met Lys Gly Phe Thr Cy - #s His His Val Val Arg    #              10605    - Leu Leu Gly Val Val Ser Lys Gly Gln Pro Th - #r Leu Val Val Met Glu    #          10750    - Leu Met Ala His Gly Asp Leu Lys Ser Tyr Le - #u Arg Ser Leu Arg Pro    #      10905    - Glu Ala Glu Asn Asn Pro Gly Arg Pro Pro Pr - #o Thr Leu Gln Glu Met    #  11050    - Ile Gln Met Ala Ala Glu Ile Ala Asp Gly Me - #t Ala Tyr Leu Asn Ala    #               11251115 - #                1120    - Lys Lys Phe Val His Arg Asp Leu Ala Ala Ar - #g Asn Cys Met Val Ala    #              11405    - His Asp Phe Thr Val Lys Ile Gly Asp Phe Gl - #y Met Thr Arg Asp Ile    #          11550    - Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Ly - #s Gly Leu Leu Pro Val    #      11705    - Arg Trp Met Ala Pro Glu Ser Leu Lys Asp Gl - #y Val Phe Thr Thr Ser    #  11850    - Ser Asp Met Trp Ser Phe Gly Val Val Leu Tr - #p Glu Ile Thr Ser Leu    #               12051195 - #                1200    - Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Gl - #u Gln Val Leu Lys Phe    #              12205    - Val Met Asp Gly Gly Tyr Leu Asp Gln Pro As - #p Asn Cys Pro Glu Arg    #          12350    - Val Thr Asp Leu Met Arg Met Cys Trp Gln Ph - #e Asn Pro Lys Met Arg    #      12505    - Pro Thr Phe Leu Glu Ile Val Asn Leu Leu Ly - #s Asp Asp Leu His Pro    #  12650    - Ser Phe Pro Glu Val Ser Phe Phe His Ser Gl - #u Glu Asn Lys Ala Pro    #               12851275 - #                1280    - Glu Ser Glu Glu Leu Glu Met Glu Phe Glu As - #p Met Glu Asn Val Pro    #              13005    - Leu Asp Arg Ser Ser His Cys Gln Arg Glu Gl - #u Ala Gly Gly Arg Asp    #          13150    - Gly Gly Ser Ser Leu Gly Phe Lys Arg Ser Ty - #r Glu Glu His Ile Pro    #      13305    - Tyr Thr His Met Asn Gly Gly Lys Lys Asn Gl - #y Arg Ile Leu Thr Leu    #  13450    - Pro Arg Ser Asn Pro Ser    1350                1355    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #               25 ATGT GCTGG    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #                24GTCA GAAT    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Probe"(A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    # 19               TGT    __________________________________________________________________________

I claim:
 1. A mutant human insulin receptor DNA, wherein the basesequence encoding Thr⁸³¹ in human insulin receptor DNA has been replacedby a base sequence encoding Ala and/or the base sequence encodingTyr¹³³⁴ therein has been replaced by a base sequence encoding Cys, or afragment of said mutant human insulin receptor DNA containing themutation, comprising the base sequence encoding Ala and/or the basesequence encoding Cys.
 2. The mutant human insulin receptor DNA asclaimed in claim 1, wherein the base sequence (ACG) encoding Thr⁸³¹ inthe exon 13 of the β-subunit in human insulin receptor DNA has beenreplaced by a base sequence (GCG) encoding Ala, or a fragment of thesame containing the mutation.
 3. The mutant human insulin receptor DNAas claimed in claim 1, wherein the base sequence (TAC) encoding Tyr¹³³⁴in the exon 22 of the β-subunit in human insulin receptor DNA has beenreplaced by a base sequence (TGC) encoding Cys, or a fragment of thesame containing the mutation.
 4. The mutant human insulin receptor DNAas claimed in claim 1 which does not bind to phosphatidylinositol3-kinase or a fragment of the same containing the mutation.
 5. The DNAwhich is complementary to a mutant human insulin receptor DNA as claimedin claim 1 or a fragment of the same containing the mutation.
 6. Adiagnostic probe for non-insulin-dependent diabetes mellitus whichcomprises a mutant human insulin receptor fragment as claimed in any oneof claims 1 to
 5. 7. A diagnostic drug for non-insulin-dependentdiabetes mellitus which contains a mutant human insulin receptorfragment as claimed in any one of claims 1 to
 5. 8. The probe of claim6, wherein said fragment comprises from 10 to 50 bases.
 9. A method fordiagnosing non-insulin-dependent diabetes mellitus, comprising the stepsof:(a) amplifying a DNA region containing the base sequence encodingThr⁸³¹ and/or Tyr¹³³⁴ in human insulin receptor DNA from a nucleic acidsample obtained from a subject; and (b) detecting a mutation of the basesequence in said DNA region, wherein the mutation is such that the basesequence encoding Thr⁸³¹ has been replaced by a base sequence encodingAla and/or the base sequence encoding Tyr¹³³⁴ has been replaced by abase sequence encoding Cys and wherein the mutation is diagnostic fornon-insulin-dependent diabetes mellitus.
 10. The method of claim 9,wherein the method for detecting the mutation is a restriction fragmentlength polymorphism (RFLP) method.
 11. The method of claim 9, whereinthe method for detecting the mutation is allele-specific hybridizationmethod.